Among the stages of the HIV life cycle, viral entry is an attractive target for the design of new therapeutic agents. The principle purpose of this project is to design and characterize an efficient entry inhibitor. Additionally we have attempted to define the basic biochemical requirements necessary for efficient inhibition of HIV-1 entry into primary CD4+ T-cells. Such information should be generally useful in the design of entry inhibitors. Soluble (s) CD4, the first entry inhibitor to be tested clinically, failed primarily because it demonstrated poor neutralizing activity against primary isolates of HIV-1. In addition, sCD4 manifested an intrinsic capacity to enhance viral replication at low concentrations. To overcome the deficiencies associated with monomeric sCD4 we fused the envelope binding domain of CD4 (D1D2) to Ig-alpha-tp creating a recombinant protein we term D1D2-Ig-alpha-tp . This recombinant protein exhibited a mass greater than 650 kilodaltons (kDa) and present twelve gp120 binding sites. The presentation of twelve closely spaced gp120 binding sites should improve the capacity of sCD4 to compete with membrane CD4 for virion associated gp120. In addition, the extremely large size of this protein should preclude any interaction between activated virion spikes and CCR5, the important co-receptor for most primary HIV infections, on the target cell membrane. We have expressed and characterized this protein in vitro and have found that it exhibits an extraordinarily high affinity for the envelopes of primary HIV-1 isolates. Importantly it efficiently inhibits the replication of virus from HIV-1 positive patient PBMCs. In viral neutralization assays D1D2-Ig-alpha-tp inhibits viral replication at levels comparable to the most potent neutralizing antibodies available. Moreover, unlike monomeric sCD4, at suboptimal concentrations this recombinant protein show no entry-enhancing activity. Each of these activities suggest that we have progressed significantly in our principle goals. Based on these observations we are evaluating D1D2-Ig-alpha-tp as a potential therapeutic agent and a potential adjuvant vaccine strategy.